Medical imaging modalities, such as MRI, X-ray, gamma scintigraphy, and CT scanning, have become extremely important tools in the diagnosis and treatment of illnesses. Some imaging of internal parts relies on inherent attributes of those parts, such as bones, to be differentiated from surrounding tissue in a particular type of imaging, such as X-ray. Other organs and anatomical components are only visible when they are specifically highlighted by particular imaging techniques.
One such technique with potential to provide images of a wide variety of anatomical components involves biotargeting image-enhancing metals. Such a procedure has the possibility of creating or enhancing images of specific organs and/or tumors or other such localized sites within the body, while reducing the background and potential interference created by simultaneous highlighting of non-desired sites.
Researchers have recognized for many years that chelating various metals increases the physiologically tolerable dosage of such metals and so permits their use in vivo to enhance images of body parts (see for example C. D. Russell and A. C. Speiser, J. Nucl. Med., 21, 1086 (1988) and U.S. Pat. No. 4,647,447 (Gries et al.)). However, such simple metal chelate image enhancers, without further modification, do not generally provide any particularly significant site specificity.
The attachment of metal chelates to tissue or organ targeting macromolecules, e.g. biomolecules such as proteins, in order to produce site specific therapeutic or diagnostic agents has been widely suggested.
Many such bifunctional chelating agents, i.e. agents which by virtue of the chelant moiety and capable of strongly binding a therapeutically or diagnostically useful metal ion and by virtue of the site-specific macromolecular component are capable of selective delivery of the chelated metal ion to the body site of interest, are known or have been proposed in the literature. Thus for example even relatively early publications in the field of MRI contrast agents, such as CB-A-2169598 (Schering) and EP-A-136812 (Technicare) suggested the use as contrast agents of paramagnetic metal ion chelates of bifunctional chelants.
The attachment or chelant moieties to site-specific macromolecules has been achieved in a number of ways, for example the mixed anhydride procedure of Krejcarek et al. (Biochemical and Biophysical Research Communications 77: 581 (1977)), the cyclic anhydride procedure of Hnatowich et al. (see Science 220: 613 (1983) and elsewhere), the backbone derivatisation procedure of Meares et al. (see Anal. Biochem. 142: 68 (1984) and elsewhere--this is a technique used by Schering in EP-A-331616 to produce site specific polychelates for use as MRI or X-ray contrast agents), and the linker molecule procedure used for example by Amersham (see WO-A-85/05554) and Nycomed (see EP-A-186947 and elsewhere) to produce paramagnetic metal ion chelates of bifunctional chelants for use as MRI contrast agents.
Thus, Krejcarek et al. (supra) disclosed how polyaminopolycarboxylic acid (PAPCA) chelants, specifically DTPA (diethylenetriaminepentaacetic acid) could be conjugated to a protein, such as human serum albumin (HSA), by reaction of the triethylamine salt of the PAPCA with isobutylchloroformate (IBCF) and by reacting the IBCF-PAPCA adduct with the protein. Their aim was to attach one radioactive metal per human serum albumin molecule for the purpose of measuring biological function.
Site specific uses of various imaging techniques all require or would be enhanced by use of a multiplicity of the appropriate metal ion conjugated to a site directed macromolecule. For example, it is believed that a 50% reduction in T.sub.1 relaxation time of water protons in a target tissue is a requirement for an effective MRI contrast agent. Considering the affinity of antibodies for their antigens and the concentration of these antigens in the target tissues, it has been calculated that each antibody molecule must carry a number of paramagnetic centers to bring about these levels of T.sub.1 reduction. (See Eckelman, et al., NATO ASI Series, Series A, 152:571 (1988)).
Unger et al. in Investigative Radiology 20:693 (1985) analyzed tumor enhancement for magnetic resonance imaging using an anti-CEA monoclonal antibody conjugated with Gd-DTPA. They found no tumor enhancement when 4 Gd atoms were bound per antibody molecule, and predicted that a far greater ratio of imaging metal atoms per macromolecule would be required to be effective.
Likewise, Schreve and Aisen in Mag. Res. in Medicine 3, 336 (1986), concluded that the concentrations of paramagnetic ion which could be delivered to a tumor using the described technology would result in large doses for humans, making this approach to imaging highly limited in its use.
For site specific image enhancement however it is important that the site specificity of the tissue or organ targeting moiety of such chelates of bifunctional chelants should not be destroyed by conjugation of the chelant moiety. Where the bifunctional chelant contains only one chelant moiety this is not generally a severe problem; however when attempts have been made to produce bifunctional polychelants by conjugating several chelant moieties onto a single site-specific macromolecule, it has been found not only may the maximum achievable chelant: site-specific macromolecule ratio be relatively limited but as the ratio achieved increases the site-specificity of the resulting bifunctional polychelant decreases.
Numerous attempts have nonetheless been made to produce bifunctional polychelants with increased numbers of chelant moieties per site-specific macromolecule.
Thus Hnatowich et al. (supra) used the cyclic anhydride of the chelant DTPA to attach it to a protein.
This is a relatively simple one-step synthesis procedure which as a result has been used by many other researchers. However, due to the presence of two cyclic anhydride groups in the starting material, widespread cross-linking of the macromolecules can lead to the production of conjugates that cannot readily be characterized (see Hnatowich et al., J. Immul. Methods 65:147 (1983)). In addition, this procedure suffers from the same drawback as that for Krejcarek's mixed anhydride method in that the uncontrolled addition of more than a few chelant moieties destroys the site-specificity of the macromolecule to which they are linked. (See also Paik et al. J. Nucl. Med. 25:1158 (1983)).
In order to overcome the problems of attaching larger numbers of chelant moieties to a site specific macromolecule without destroying its site-specificity, i.e. without disturbing its binding site(s), there have been many proposals for the use of a backbone molecule to which large numbers of chelant moieties can be attached to produce a polychelant one or more of which can then be conjugated to the site-specific macromolecule to produce the bifunctional polychelant.
The by now conventional cyclic anhydride conjugation technique of Hnatowich et al. (supra) has thus been used to produce bifunctional polychelants in which the chelant moieties are residues of open chain PAPCAs, such as EDTA and DTPA, and in which the backbone molecule is a polyamine such as polylysine or polyethyleneimine. Thus for example Manabe et al. in Biochemica et Biophysica Acta 883: 460-467 (1986) reported attaching up to 105 DTPA residues onto a poly-L-lysine backbone using the cyclic anhydride method and also attaching polylysine polyDTPA polychelants onto monoclonal antibody (anti-HLA IgG.sub.1) using a 2-pyridyl disulphide linker achieving a substitution of up to about 42.5 chelants (DTPA residues) per site-specific macromolecule. Torchlin et al. in Hybridoma 6:229-240 (1987) also reported attaching DTPA and EDTA to polyethyleneimine and polylysine backbones which were then attached to a myosin-specific monoclonal antibody, or its Fab fragment, to produce bifunctional polychelants for use in MRI or scintigraphy.
While Manabe and Torchlin have reported the production of bifunctional polychelants, the cyclic anhydride route adopted by Manabe poses cross-linking and hence characterization problems and Torchlin et al in their conclusion doubted that their technique would enable the paramagnetic metal concentration to be increased sufficiently to permit MRI of tumours.
There is thus a continuing need for improved bifunctional polychelants and the present invention resides in the provision of novel and improved bifunctional polychelants, particularly such polychelants that can be produced from relatively non-complex chelant starting materials. More particularly, the present invention resides in the provision of bifunctional polychelants, and their chelates, containing macrocyclic chelant moieties, that is to say chelants which contain at least one macrocyclic structural element which serves at least in part to define the seat for the chelated ion. Macrocyclic chelants, (for example 1,4,7,10-tetraazacyclododecane tetraacetic acid) are themselves well known as chelants capable of forming very stable chelate complexes, and in particular complexes which unlike those of non-macrocyclic chelants are particularly stable kinetically as well as themodynamically, but they cannot be effectively linked to backbone molecules such as polylysine by the prior art cyclic anhydride (Hnatowich) or mixed anhydride (Krejcarek) procedures, as the procedures are presently applied.
This invention provides an efficient and successful means for creating bifunctional poly(macrocyclic chelants) (BPMCs) as well as the BPMCs and their chelates themselves. Numerous obstacles previously present in creating a biologically functional imaging molecule with a multiplicity of chelating sites have been overcome, and in particular cross linking of the polychelants has been avoided, allowing for better solubility and better site-specificity, due to the workable size of the bifunctional polychelant.